The present invention relates, in general, to a method for preparing three-dimensional porous scaffolds which can be used as supports or culture matrices for in-vitro tissue culture and, more particularly, to the use of effervescent salts in preparing scaffolds through a gel phase, thereby allowing the scaffolds to be molded in desirable forms and to have a desirable pore size and porosity.
To be used for bio-tissue culture, polymers are basically required to be of biocompatibility and biodegradability. The aliphatic polyesters which bear lactic acid or glycolic acid as a backbone unit were approved as being satisfactory to the requirement by the Food and Drug Administration (FDA), U.S.A., and most widely used now. Examples of such biocompatible and biodegradable aliphatic polyesters include poly(lactic acid) (PLA) poly(glycolic acid) (PGA), poly(D,L-lactic-co-glycolic acid) (PLGA), poly(caprolactone), poly(valerolactone), poly(hydroxybutyrate), poly(hydroxy valerate), etc.
Proven to be biocompatible, the aliphatic polyesters have been widely used as drug delivery carriers or sutures for a long period of time.
PLGA is found to afford biodegradable polymers with various degradation periods by controlling the ratio of lactic acid monomer and glycolic acid monomer and/or modifying the synthesis procedure thereof.
In addition to biodegradability and biocompatibility, other requirements for the polymers for bio-tissue culture are a surface area large enough to allow cell adhesion at high densities, a pore size large enough to enable the vascularization in the cultured tissue after transplantation into a host and the transmission of substances, such as nutrients, growth factors and hormones, and the interconnectivity of the pores.
Typically, the porous polymeric scaffolds fulfilling the above requirements are prepared as follows.
The most popular and commercially available are scaffolds consisting of PGA sutures (unwoven PGA fiber mesh). They are made in three-dimensional shapes by thermally treating randomly entangled threads of suture. The mesh exhibits very high porosity and sufficiently large pore size in addition to being of high interconnectivity, but finds a limited range of applications on account of poor mechanical strength (see: A. G. Mikos, Y. Bao, L. G. Cima, D. E. Ingber, J. P. Vacanti, and R. Langer, J. Biomed. Mater. Res. (1993) 27, 183-189).
Another preparing method of the porous polymeric scaffolds is of particulate leaching, favored by A. G. Mikos et al. (See: A. G. Mikos, G. Sarakinos, S. M. Leite, J. P. Vacanti, and R. Langer, Biomaterials (1993) 14, 5, 323-330; A. G. Mikos, A. J. Thorsen, L. A. Czerwonka, Y. Bao, R, Langer, D. N. Winslow, and J. P. Vacanti, Polymer (1994) 35, 5, 1068-1077). The particulate leaching method has an advantage of easily controlling pore sizes of the scaffolds in dependence on the size of the salt (NaCl) employed, but suffers from a disadvantage in that salts remaining in the scaffolds or their rough morphology cause cell damage.
Besides, an emulsion freeze-drying method and a high pressure gas expansion method can be used for the preparation of such scaffolds (see: k. Whang, C. H. Thomas, K. E. Healy, G. Nuber, Polymer (1995) 36, 4, 837-842; J. J. Mooney, D. F. Baldwin, N. P. Suh, J. P. Vacanti, and R. Langer, Biomaterials (1996) 17, 1417-1422). Despite their own advantages, the methods have the limitation of there being difficulties in making open cellular pores.
In recent, attempts have been made to construct the scaffolds by taking advantage of the phase separation of polymer solutions (H. Lo, M. S. Ponticiello, K. W. Leong, Tissue Eng. (1995) 1, 15-28; H. Lo, S. Kadiyala, S. e. Guggino, K. W. Leong, J. Biomed. Mater. Res. (1996) 30, 475-484; Ch. Schugens, V. Maguet., Ch, Grandfils, R. Jerome, Ph. Teyssie, J. Biomed. Mater. Res. (1996) 30, 449-461).
As mentioned above, various methods have been developed for the preparation of three-dimensional polymeric scaffolds in which cell adhesion and differentiation can be induced. Nevertheless, there remain problems to be solved in preparing three-dimensional scaffolds for tissue culture with biodegradable polymers. At present, only a few companies, such as Advanced Tissue Science Inc. and Texas Biotechnology Inc. have been successful in the commercialization of such scaffolds, wherein PGA suture is utilized on a small scale.
It is an object of the present invention to overcome the above problems encountered in prior arts and to provide a method for preparing biodegradable, three-dimensional, porous scaffolds for tissue culture, whereby the scaffolds can be molded in desirable forms and have desirable pore sizes and porosities.
Based on the present invention, the above object could be accomplished by a provision of a method for preparing biodegradable, three-dimensional, porous scaffolds for tissue culture, comprising the steps of dissolving a biodegradable polymer in an organic solvent to prepare a polymeric solution of high viscosity, homogeneously mixing an effervescent salt in the polymeric solution to give a polymer/salt/organic solvent mixed gel, removing the organic solvent from the polymer/salt/organic solvent mixed gel, submerging the organic solvent-free polymer/salt gel slurry in a hot aqueous solution or acidic solution to render the salt to effervesce at room temperature to afford a three-dimensional polymeric structure, and washing with distilled water and freeze-drying the polymeric structure.